Cells of the immature nucleus pulposus (NP) are biologically active and play an important role in regulating matrix biosynthesis during intervertebral disc (IVD) development and aging. The immature NP contains a large amount of highly vacuolated cells of notochordal origin that undergo early morphological changes with age. Cells of the immature NP, but not mature NP, have been shown to enhance matrix synthesis in multiple cell types and to attenuate degeneration in animal models. This unique biosynthetic effect may be due to the presence of a large amount of notochordal cells present in the immature NP which subsequently disappear with aging in mature NP. Thus, there is great interest in identifying the existence of, and unique features of a distinct notochordal-like cell population in the NP. We have developed a fluorescence-activated cell sorting (FACS) protocol (based on cell size and auto-fluorescence) that identifies a unique population of notochordal-like NP cells with a distinct morphological appearance and molecular phenotype (i.e. unique integrin and mRNA expression pattern) that is distinct from other cell types in the NP and AF of animal tissues. We propose to use this method to identify a notochordal-like NP cell population within the human NP based on expression of unique molecular markers identified here. The central hypothesis of this proposal is that notochordal-like NP cells are a metabolically and phenotypically distinct NP cell population, with unique expression patterns for cell surface receptors, matrix and secreted proteins that are altered upon aging. We propose to select or enrich notochordal-like NP cells by these unique cell surface receptors and study their biological behaviors and the potential for regeneration of NP-like matrix in vitro. In Aim 1, a unique notochordal-like NP cell population will be identified in the rat using FACS based on the distinct morphology in order to define the unique expression of cell surface receptors (i.e., integrin subunits and select CD antigens) for this population. Biological behaviors of cell survival, adhesion, morphology, and secreted protein synthesis will also be examined for 2D or 3D culture of these unique cells in vitro. Differences in molecular phenotype and biological behaviors from immature to aged rat will be used to test for the presence of a unique immature cell phenotype and its changes with age. In Aim 2, the existence of a unique notochordal-like NP cell phenotype in the juvenile and adult human IVD will be evaluated based on expression of a unique set of markers determined in the notochordal- like NP cells of the rat. A unique expression profile will first be confirmed by immunostaining and flow cytometry in the human, followed NP cell sorting to yield a unique notochordal-like NP cell subpopulation. Biological behaviors of cell survival, adhesion, morphology, secreted protein synthesis, and the extracellular matrix regeneration potential will also be examined for 2D or 3D culture of these unique FACS-sorted NP cells in vitro. Results from the study will reveal the existence of, and precisely characterize the molecular features and biological behaviors of an NP cell subpopulation with hypothesized potential to regulate disc regeneration. This work will build a foundation for multiple paths to clinical translation of autologous NP cell and precursor cell therapies for the treatment of disc pathology, based on the development of new protocols and technologies designed to promote healthy NP cell survival, and matrix synthesis. PUBLIC HEALTH RELEVANCE: Intervertebral disc degenerative disorders and resulting pain may be associated with aging- or pathology- related cell loss, and changes in cell phenotype. Cellular therapies for biologically-driven regeneration of intervertebral disc are of great interest, whereby disc cells from human patients or progenitor cells are used to generate new matrix in vitro or following re-implantation in vivo. This project will use a cell sorting technology to identify a population of disc cells which retain characteristics of an immature cell phenotype, and to investigate the key contributors or therapeutic targets for maintaining this immature cell-like phenotype and matrix synthesis during cell regeneration.